Journal: The Journal of Experimental Medicine

Article Title: Subspecialization of Cxcr5 + T Cells

doi:

Figure Lengend Snippet: Effector machinery of CD57 +/ −CD45RA +/ − or CD57 +/ − CXCR5 +/ − CD4 T cells. (A) Intracellular cytokine analyses of TNF-α, IL-2, IL-10, IFN-γ, and IL-4. (B) ELISA of IL-10 and IL-4. Isolated T cells were activated with 50 ng/ml PMA and 1 μg/ml ionomycin for 4 h for intracellular cytokine analyses, or 24 h for ELISA of IL-10 and IL-4. Representatives of at least four independent experiments are shown.

Article Snippet: In brief, acetone-fixed sections were stained with the primary monoclonal antibody to CXCR5 (R&D Systems), followed by incubation in biotinylated goat anti–mouse IgG (Vector Laboratories).

Techniques: Enzyme-linked Immunosorbent Assay, Isolation

Journal: The Journal of Experimental Medicine

Article Title: Subspecialization of Cxcr5 + T Cells

doi:

Figure Lengend Snippet: Identification of CD57 + CXCR5 + T cells and their localization in GCs. (A) Flow analyses of CD57 and CXCR5 expression on tonsil CD4 T cells. (B) Specific localization of CD57 + CD4 T cells in GCs. (C) Localization of CD57 + CXCR5 + CD4 T cells in GCs. Antibodies to CD57 (green), CD4 (blue), and IgD (red; B) or CXCR5 (red; C) were used for in situ immunohistochemistry.

Article Snippet: In brief, acetone-fixed sections were stained with the primary monoclonal antibody to CXCR5 (R&D Systems), followed by incubation in biotinylated goat anti–mouse IgG (Vector Laboratories).

Techniques: Expressing, In Situ, Immunohistochemistry

Journal: The Journal of Experimental Medicine

Article Title: Subspecialization of Cxcr5 + T Cells

doi:

Figure Lengend Snippet: Chemotactic responses (A) and chemokine receptor expression (B) by CD57 +/2 CXCR5 +/ − CD4 T cells. Optimal concentrations of 5 μg/ml BLC, 1 μg/ml ELC, and 100 ng/ml SDF-1 were used for chemotaxis experiments. Freshly isolated tonsil cells were used for chemotaxis and flow analyses of tonsil CD4 T cell subsets. Representatives of three independent experiments are shown.

Article Snippet: In brief, acetone-fixed sections were stained with the primary monoclonal antibody to CXCR5 (R&D Systems), followed by incubation in biotinylated goat anti–mouse IgG (Vector Laboratories).

Techniques: Expressing, Chemotaxis Assay, Isolation

Journal: The Journal of Experimental Medicine

Article Title: Subspecialization of Cxcr5 + T Cells

doi:

Figure Lengend Snippet: Spontaneous B cell helper activity of CD57 +/ − CXCR5 +/ − CD4 T cells. Sorted CD57 + CXCR5 + , CD57 − CXCR5 + , and CXCR5 − CD4 T cells were cocultured with B cells from the same tonsil for 11–13 d in the absence of any stimulatory agents followed by analyses of secreted IgG, IgA, and IgM. Representatives of at least three independent experiments are shown.

Article Snippet: In brief, acetone-fixed sections were stained with the primary monoclonal antibody to CXCR5 (R&D Systems), followed by incubation in biotinylated goat anti–mouse IgG (Vector Laboratories).

Techniques: Activity Assay

Journal: The Journal of Experimental Medicine

Article Title: Subspecialization of Cxcr5 + T Cells

doi:

Figure Lengend Snippet: Phenotype and effector function of circulating CXCR5 + T cells. (A) Surface phenotype of circulating naive or CXCR5 +/ − memory T cells. **Significant differences between CXCR5 + and CXCR5 − memory cells. (B) IL-4/IFN-γ production capabilities of CXCR5 +/ − T cells during repeated T cell receptor activation (each cycle is composed of 4-d activation with anti-CD3 and anti-CD28 followed by 3-d resting in the presence of IL-2). (C) B cell help activity of circulating CXCR5 + T cells after T cell receptor activation. Naive (IgD + ) or memory (IgD − ) B cells (2 × 10 4 ) from peripheral blood were cultured for 14 d in the presence or absence of various numbers (10 3 , 5 × 10 3 , 10 4 , 2.5 × 10 4 , 5 × 10 4 , and 10 5 ) of autologous T cells (CXCR5 − CD45RA + , CXCR5 + CD45RA − , or CXCR5 − CD45RA − ). Concentrations of IgG, IgA, and IgM in the culture supernatants were measured by ELISA. T cell numbers required for peak levels of antibody production varied among donors or experiments. One peak value with the best antibody production in each T cell group is shown. (D) Loss of CXCR5 expression during T cell receptor activation with anti-CD3 and anti-CD28. Error bars indicate SD of results from at least five different experiments (A). Representatives of three independent experiments are shown (B and D), and results from seven different donors (C) are shown.

Article Snippet: In brief, acetone-fixed sections were stained with the primary monoclonal antibody to CXCR5 (R&D Systems), followed by incubation in biotinylated goat anti–mouse IgG (Vector Laboratories).

Techniques: Activation Assay, Activity Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Journal of Extracellular Vesicles

Article Title: Bioorthogonally surface‐edited extracellular vesicles based on metabolic glycoengineering for CD44‐mediated targeting of inflammatory diseases

doi: 10.1002/jev2.12077

Figure Lengend Snippet: In vivo biodistribution of PHA‐EVs in inflammatory animal models with tumour and RA. (a) In vivo fluorescence images of Cy5.5‐EVs and Cy5.5‐PHA‐EVs in CIA mice. (b) Fluorescence intensity of Cy5.5‐EVs and Cy5.5‐PHA‐EVs in the inflamed joints of CIA mice as a function of time ( n = 3). (c) Distribution of CD44 and EV in the inflamed synovium of CIA mice. (d) Whole‐body fluorescence images of Cy5.5‐EVs and Cy5.5‐PHA‐EVs in PC3 tumour‐bearing mice. (e) Fluorescence intensity of Cy5.5‐EVs and Cy5.5‐PHA‐EVs in the tumour site as a function of time ( n = 3). (f) Distribution of CD44 and EV in tumour tissue. (b and e) * P < 0.05, analyzed by two‐way ANOVA. Error bars represent standard error

Article Snippet: Cy5.5 NHS ester dye was purchased from GE Healthcare (IL, USA).

Techniques: In Vivo, Fluorescence

Journal: Journal of Korean Neurosurgical Society

Article Title: In Vivo Spinal Distribution of Cy5.5 Fluorescent Dye after Injection via the Lateral Ventricle and Cisterna Magna in Rat Model

doi: 10.3340/jkns.2017.0252

Figure Lengend Snippet: Surgical procedures for LV and CM injection. Surgical procedures for LV and CM injection were shown in (A) and (B), respectively. A : For LV injection, after anesthesia, rats were fixed with a stereotaxic device and cut to expose the bregma. After making a hole with a drill, a needle was injected into the LV. B : For CM injection, after anesthesia, rats were fixed with a stereotaxic device. After surgery to expose CM, Cy5.5 was injected into CM. C : Each region of interest to measure Cy5.5 was demonstrated. H : head, C : cervical, T : thoracic, L : lumbar, LV : lateral ventricle, CM : cisterna magna.

Article Snippet: 20 nM Cy5.5 (Amersham CyDye™ mono-reactive NHS Ester, GE Healthcare, Piscataway, NJ, USA) in 20 μL saline was injected using a syringe pump (LEGATO™111, KD scientific, Holliston, MA, USA).

Techniques: Injection

Journal: Journal of Korean Neurosurgical Society

Article Title: In Vivo Spinal Distribution of Cy5.5 Fluorescent Dye after Injection via the Lateral Ventricle and Cisterna Magna in Rat Model

doi: 10.3340/jkns.2017.0252

Figure Lengend Snippet: Optical imaging of Cy5.5 after LV and CM injection. The total flux of Cy5.5 signal at each region of interest was evaluated at 12, 24, 48, and 72 hours after injection of Cy5.5 via LV and CM route. A : In the LV injection the signal of Cy5.5 was observed in the thoracic and lumbar region within 12 hours. B : In the CM injection, most Cy5.5 signal remained in the head region until 72 hours. LV : lateral ventricle, CM : cisterna magna.

Article Snippet: 20 nM Cy5.5 (Amersham CyDye™ mono-reactive NHS Ester, GE Healthcare, Piscataway, NJ, USA) in 20 μL saline was injected using a syringe pump (LEGATO™111, KD scientific, Holliston, MA, USA).

Techniques: Optical Imaging, Injection

Journal: Journal of Korean Neurosurgical Society

Article Title: In Vivo Spinal Distribution of Cy5.5 Fluorescent Dye after Injection via the Lateral Ventricle and Cisterna Magna in Rat Model

doi: 10.3340/jkns.2017.0252

Figure Lengend Snippet: Quantitation of the in vivo signal intensity of Cy5.5 after LV and CM injection. The total flux of Cy5.5 signal was quantitated at each region of interest. A : After LV injection, the sum of total fluxes was shown at each time point. B : The total flux of Cy5.5 at each region of interest was shown at various time points. C : After CM injection, the sum of total fluxes was shown at each time point. D : The total flux of Cy5.5 at each region of interest was shown at various time points. * p <0.05. LV : lateral ventricle, CM : cisterna magna.

Article Snippet: 20 nM Cy5.5 (Amersham CyDye™ mono-reactive NHS Ester, GE Healthcare, Piscataway, NJ, USA) in 20 μL saline was injected using a syringe pump (LEGATO™111, KD scientific, Holliston, MA, USA).

Techniques: Quantitation Assay, In Vivo, Injection

Journal: Journal of Korean Neurosurgical Society

Article Title: In Vivo Spinal Distribution of Cy5.5 Fluorescent Dye after Injection via the Lateral Ventricle and Cisterna Magna in Rat Model

doi: 10.3340/jkns.2017.0252

Figure Lengend Snippet: Comparison of the in vivo signal intensity of Cy5.5 according to the injection route at each region of interest. The regional total flux of Cy5.5 at each time point was compared between LV and CM injection route. A : In head region, CM injection showed slightly higher signal intensity than that of the LV injection but not statistically significant. B : In cervical region, both injection methods showed similar signal intensity. C and D : In thoracic and lumbar region, LV injection showed significantly higher signal intensity than CM injection. * p <0.05. LV : lateral ventricle, CM : cisterna magna.

Article Snippet: 20 nM Cy5.5 (Amersham CyDye™ mono-reactive NHS Ester, GE Healthcare, Piscataway, NJ, USA) in 20 μL saline was injected using a syringe pump (LEGATO™111, KD scientific, Holliston, MA, USA).

Techniques: In Vivo, Injection

Journal: bioRxiv

Article Title: Multi-modal Single-molecule Imaging with Continuously Controlled Spectral-resolution (CoCoS) Microscopy

doi: 10.1101/2020.10.13.330910

Figure Lengend Snippet: Resolving the shades of red dyes. a) schematic illustration explaining the procedure to obtain spectral information with CoCoS imaging. First, a localization image is captured using RPA of 180°, then the prisms are rotated to the optimal RPA for the spectral resolution of the imaged dyes (here 120°) and a second image is captured. An intensity profile of a molecule in the spectral image, with respect to the molecule’s coordinates in the localization image, contains the emission spectrum of the molecule. b) Three different red fluorophores were used to label 100 nm silica beads. The beads were mixed and imaged according to the procedure illustrated in a, shown in the false colored FOV image on the left. The localization image presented in white is overlaid on the spectral image in red, showing the dispersed molecular spectra. On the right, the localization image is shown after classification of the dyes according to their spectra using cross-correlation of the individual spectrum with average dye spectra (see methods). Blue, yellow and red refer to CF640R, AF647 and Cy 5.5 respectively. Colored dashed squares mark the molecules presented in panel c. Scale bars are 30 µm. c) Normalized spectral intensity profile of three selected molecules in panel b. Cropped images of the molecules after alignment and Gaussian filtering of the spectral image is shown at the bottom. Dashed lines indicate the localization coordinate. d) An overlay of all individual spectral profiles extracted from the FOV. Mean cross-correlation scores used for the classification of the spectra are shown in the legend. Vertical lines show the theoretical emission maxima of the corresponding dyes’ spectra, showing that molecular spectra with maxima differing by only ~6nm are resolvable with CoCoS.

Article Snippet: For the far-red beads experiment the same procedure was followed mixing a single 75 µM DBCO/BCN activated dye (AF647-DBCO, Cy5.5-DBCO, Jena Bioscience, Germany; CF640R-BCN, Biotium, USA) with the azide-covered nano-beads to create three different stock solutions.

Techniques: Imaging

Journal: Journal of Nanobiotechnology

Article Title: Neutrophil membrane-coated and MMP2-responsive nanoparticles deliver PIM1 inhibitor to alleviate inflammatory arthritis through inhibiting Th17 cell differentiation

doi: 10.1186/s12951-026-04280-x

Figure Lengend Snippet: Preparation and characterization of NM@MRP-NP ( A ) Morphology of MRP-NP, NM and NM@MRP-NP observed by SEM. Scale bar = 100 nm. ( B - D ) Size distributions ( B ), Z-average diameter ( C ) and zeta potential ( D ) of MRP-NP, NM and NM@MRP-NP measured by DLS ( n = 5). ( E ) Z-average diameter of MRP-NP, NM and NM@MRP-NP after incubation with mouse serum or FBS for different durations ( n = 5). ( F ) Fluorescence image of NM@MRP-NP loaded with different concentrations of Cy5.5. ( G ) Drug release rates of MRP-NP and NM@MRP-NP with or without MMP2 ( n = 5)

Article Snippet: The resulting NM@MRP-NP was collected by centrifugation at 20,000×g at 4 °C for 20 min. For the fluorescence tracing experiment, Cy5.5 (MCE, #HY-D0924) was substituted for AZD1208 and encapsulated into the nanoparticles.

Techniques: Zeta Potential Analyzer, Incubation, Fluorescence

Journal: Journal of Nanobiotechnology

Article Title: Neutrophil membrane-coated and MMP2-responsive nanoparticles deliver PIM1 inhibitor to alleviate inflammatory arthritis through inhibiting Th17 cell differentiation

doi: 10.1186/s12951-026-04280-x

Figure Lengend Snippet: Biological characteristics of NM@MRP-NP. ( A ) Western blot analysis of the expression of CD11a, CD11b and L-selectin on the neutrophil membrane before and after LPS treatment ( n = 9). ( B ) Representative immunofluorescence images of CD11a, CD11b and L-selectin on the neutrophil membrane before and after LPS treatment. Scale bar = 20 μm. ( C - D ) Western blot analysis ( C ) and immunofluorescence staining ( D ) confirmed the transfer of CD11a, CD11b and L-selectin to the NM@MRP-NP. Scale bar = 100 μm. ( E - F ) Uptake of Cy5.5 by activated or unactivated CD4 + T cells treated with vehicle, MRP-NP, NM@MRP-NP or NM@MRP-NP+MMP2 ( n = 9). The statistical analyses were performed as follows: paired t test (A, F) and one-way ANOVA followed by Bonferroni’s post hoc comparisons test ( F ). ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001, and n.s. indicates no statistical significance

Article Snippet: The resulting NM@MRP-NP was collected by centrifugation at 20,000×g at 4 °C for 20 min. For the fluorescence tracing experiment, Cy5.5 (MCE, #HY-D0924) was substituted for AZD1208 and encapsulated into the nanoparticles.

Techniques: Western Blot, Expressing, Membrane, Immunofluorescence, Staining

Journal: Journal of Nanobiotechnology

Article Title: Neutrophil membrane-coated and MMP2-responsive nanoparticles deliver PIM1 inhibitor to alleviate inflammatory arthritis through inhibiting Th17 cell differentiation

doi: 10.1186/s12951-026-04280-x

Figure Lengend Snippet: Targeting effect of NM@MRP-NP in vivo. ( A ) Cy5.5 fluorescence images of the inflamed ankles of SKG mice at different time points after treatment with vehicle, free Cy5.5, Cy5.5-labeled MRP-NP or NM@MRP-NP. ( B ) Cy5.5 fluorescence images of synovial fluid collected from SKG mice 8 h after treatment with vehicle, free Cy5.5, Cy5.5-labeled MRP-NP or NM@MRP-NP. ( C ) Cy5.5 fluorescence images of the ankles and the major organs dissected from SKG mice 8 h after treatment with vehicle, free Cy5.5, Cy5.5-labeled MRP-NP or NM@MRP-NP. ( D ) Immunofluorescence images of Cy5.5 and CD4 + T cells in the inflamed joints of SKG mice treated with vehicle, free Cy5.5, Cy5.5-labeled MRP-NP or NM@MRP-NP. Scale bar = 50 μm. ( E ) Quantitative analysis of the Cy5.5 fluorescent signal in the inflamed ankles of SKG mice at different time points after receiving Cy5.5-labeled NM@MRP-NP ( n = 6). ( F ) Quantitative analysis of Cy5.5 fluorescent signal in synovial fluid collected from SKG mice 8 h after treatment with vehicle, free Cy5.5, Cy5.5-labeled MRP-NP or NM@MRP-NP ( n = 5). ( G ) Quantitative analysis of Cy5.5 fluorescence signal in the ankles and the major organs dissected from SKG mice 8 h after treatment with vehicle, free Cy5.5, Cy5.5-labeled MRP-NP or NM@MRP-NP ( n = 5). ( H ) Line-scan analysis of the colocalization of Cy5.5 and CD4 + T cells in the immunofluorescence images in Fig. 7D. The statistical analyses were performed as follows: one-way ANOVA followed by Bonferroni’s post hoc comparisons test (F, G). **** indicates p < 0.0001

Article Snippet: The resulting NM@MRP-NP was collected by centrifugation at 20,000×g at 4 °C for 20 min. For the fluorescence tracing experiment, Cy5.5 (MCE, #HY-D0924) was substituted for AZD1208 and encapsulated into the nanoparticles.

Techniques: In Vivo, Fluorescence, Labeling, Immunofluorescence